Liver toxicity is a major barrier to the FDA approval of many new drugs. Moreover, since the liver is the major site of metabolism of most xenobiotics, it is essential to know how the liver metabolizes a potential new drug. Because of the many species differences between humans and commonly used animal models, testing on human hepatocytes remains the gold standard for drug screening. However, because the source of these hepatocytes is usually from livers that have been declined for use in transplantation, typically because of extended ischemic time, the supply of high quality cells is severely limited. The long-term goal of this project is to reliably improve the availability and viability of primary human hepatocytes isolated from non-transplantable Donation after Cardiac Death (DCD) livers through the modification of a hypothermic machine perfusion (HMP) resuscitation system that we have developed to restore function to ischemia damaged DCD livers for use in transplantation. Human hepatocytes are typically isolated from the pool of non-transplantable DCD organs which has been estimated to be as high as 40% of the current donor pool. However, inconsistent yield and quality of the isolated hepatocytes from these donors has led to limited availability resulting in higher costs for these cells. Major factors contributing to this inconsistency are the extended periods of warm ischemia experienced by these organs and the preservation process (simple cold storage) which does not resuscitate these organs. The HepatoSys solution (HS) coupled with HMP was developed to restore depleted energy stores, maintain membrane stability, and minimize oxidation damage resulting in a resuscitated liver. Our results indicate that our process can be used to successfully isolate high quality hepatocytes from otherwise unusable livers. In addition, we will add further commercial value to the technology by testing prolonged HMP of livers to provide greater flexibility in making fresh cells available, and the ability of additional oxygenated cold storage of hepatocytes in the HS to improve the quality of freshly isolated and cryopreserved hepatocytes. To achieve these goals, we propose the following aims. Specific Aim 1: Test the efficacy of prolonged HMP of livers with the HepatoSys solution on yield, viability, plateability and function of fresh hepatocytes isolated from rat DCD livers. Test the effect of up to 48 hours HMP followed by hepatocyte isolation on yield, viability, plateability and function of freshly plated isolated hepatocytes from DCD rats. Specific Aim 2: Test the efficacy of additional oxygenated cold storage (CS) of fresh hepatocytes after isolation in the HepatoSys solution on yield, viability, plateability and functio from DCD rat livers Test the ability of additional CS of isolated hepatocytes in the HS from DCD rats to maintain or improve the viability, plateability of freshly isolated rat hepatocytes. Specifc Aim 3: Test the efficacy of optimized conditions identified in aims 1 and 2 on yield, viability, plateability and function of freshly isolated hepatocytes from a small group of human DCD livers. Completion of these specific aims will provide a proof of concept that this technology has a potentially commercially valuable method to obtain viable, plateable and functional hepatocytes from livers currently not usable while providing greater flexibility in making freshly isolated hepatocytes available which will allow TRL and HepatoSys to increase market share. A Phase II application will seek to add further commercial value to the technology by expanding the pool to even poorer quality donor livers and improving the quality of cryopreserved hepatocytes.